Emerging therapies have begun to evaluate the abilities of Müller glial cells (MGCs) to protect and/or regenerate neurons following retina injury. The migration of donor cells is central to many reparative strategies, where cells must achieve appropriate positioning to facilitate localized repair. Although chemical cues have been implicated in the MGC migratory responses of numerous retinopathies, MGC-based therapies have yet to explore the extent to which external biochemical stimuli can direct MGC behavior. The current study uses a microfluidics-based assay to evaluate the migration of cultured rMC-1 cells (as model MGC) in response to quantitatively-controlled microenvironments of signaling factors implicated in retinal regeneration: basic Fibroblast Growth factor (bFGF or FGF2); Fibroblast Growth factor 8 (FGF8); Vascular Endothelial Growth Factor (VEGF); and Epidermal Growth Factor (EGF). Findings indicate that rMC-1 cells exhibited minimal motility in response to FGF2, FGF8 and VEGF, but highly-directional migration in response to EGF. Further, the responses were blocked by inhibitors of EGF-R and of the MAPK signaling pathway. Significantly, microfluidics data demonstrate that changes in the EGF gradient (i.e. change in EGF concentration over distance) resulted in the directional chemotactic migration of the cells. By contrast, small increases in EGF concentration, alone, resulted in non-directional cell motility, or chemokinesis. This microfluidics-enhanced approach, incorporating the ability both to modulate and asses the responses of motile donor cells to a range of potential chemotactic stimuli, can be applied to potential donor cell populations obtained directly from human specimens, and readily expanded to incorporate drug-eluting biomaterials and combinations of desired ligands.